Lysine 77 is a key residue in aggregation of equinatoxin II, a pore-forming toxin from sea anemone Actinia equina

Among eighteen point mutants of equinatoxin II produced in E. coli, containing a single cystein substitution at variable position, EqtIIK77C was chosen for its peculiar properties. It was almost 100 times less hemolytic than the wild-type, but its hemolytic activity could be restored by chemical mod...

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Published inThe Journal of membrane biology Vol. 173; no. 1; pp. 47 - 55
Main Authors Anderluh, G, Barlic, A, Potrich, C, Macek, P, Menestrina, G
Format Journal Article
LanguageEnglish
Published United States 01.01.2000
Subjects
Amino Acid Substitution
Animals
Binding Sites
Cnidarian Venoms - blood
Cnidarian Venoms - chemistry
Cnidarian Venoms - toxicity
Cytotoxins - blood
Cytotoxins - chemistry
Cytotoxins - toxicity
Erythrocyte Membrane - drug effects
Erythrocytes - drug effects
Escherichia coli
Hemolysis
Humans
In Vitro Techniques
Kinetics
Lysine
Mutagenesis, Site-Directed
Point Mutation
Recombinant Proteins - blood
Recombinant Proteins - chemistry
Recombinant Proteins - toxicity
Sea Anemones
Spectroscopy, Fourier Transform Infrared
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Summary:Among eighteen point mutants of equinatoxin II produced in E. coli, containing a single cystein substitution at variable position, EqtIIK77C was chosen for its peculiar properties. It was almost 100 times less hemolytic than the wild-type, but its hemolytic activity could be restored by chemical modification of the thiol group, provided that a positive charge was reintroduced. This indicates that a positive charge at this position is necessary for toxin activity. The mutant formed larger pores as compared to the wild type, but displayed the same cation selectivity. The pores reverted to normal size upon reintroduction of the positive charge. The conformation of EqtIIK77C and its binding to lipid membranes, either vesicles or red blood cells, was almost normal. However the kinetics of calcein release from lipid vesicles was substantially slower than that of the wild-type. Taken together with the different size of the pore formed, this is an indication that mutation of Lys77 --> Cys influences the normal development of the aggregate which is required for assembling the functional pore.
ISSN:0022-2631
1432-1424
DOI:10.1007/s002320001006